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Long non-coding RNA XIST regulates PTEN expression by sponging miR-181a and promotes hepatocellular carcinoma progression

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ABSTRACT

Background: Tumor metastasis often occurs in hepatocellular carcinoma (HCC) and influences the patient’s prognosis, and microRNAs are reported to play key roles in tumor metastasis. This study was conducted to explore the effect of microRNAs on HCC metastasis.

Methods: The levels of miR-181a in HCC tissues, adjacent tissues, metastatic HCC tissues, and non-metastatic HCC tissues at different stages were determined by qRT-PCR. Effect of miR-181a on the proliferation, invasion, and metastasis of HCC cells was estimated by cell counting kits-8 (CCK-8), wound-healing, and Transwell assays. Software analysis and luciferase assays were used to explore the target gene of miR-181a.

Results: MiR-181a was up-regulated in HCC tissues and its expression level in metastatic HCC tissues was much higher than in non-metastasis samples. PTEN was found to be a target gene of miR-181a. MiR-181a had multiple binding sites with the long non-coding RNA (lncRNA) XIST. The regulation of miR-181a on PTEN was mediated by lncRNA XIST. The proliferation and invasion of cells with siXIST were significantly enhanced compared with those of control cells, while knockdown of miR-181a abolished the enhancing effects.

Conclusions: MiR-181a can promote HCC metastasis by targeting PTEN, which is regulated by lncRNA XIST.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3216-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


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miR-181a is regulated by lncRNA XIST. a Diagram of the three miR-181a putative binding sites and corresponding mutant sites in XIST mRNA sequences. b Effects of miR-181a on the expression of reporter genes containing wt-XIST or mut-XIST sequences. ** vs Blank, p < 0.01. c Relative expression of XIST in HCC cells and the normal liver cells. ** vs L-02, p < 0.01. d Relative expression of XIST in HCCLM3 cells after transfection with miR-181a inhibitor. ** vs Blank, p < 0.01. e Relative expression of miR-181a in Huh7 cells after transfection with si-XIST or si-Scramble. ** vs Blank, p < 0.01. f Relative expression of miR-181a in HCC clinical samples and the normal. g The relationship between relative expression of XIST and miR-181a. lncRNA, long non-coding RNA; mut, mutant; wt, wild-type; HCC, hepatocellular carcinoma
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Fig6: miR-181a is regulated by lncRNA XIST. a Diagram of the three miR-181a putative binding sites and corresponding mutant sites in XIST mRNA sequences. b Effects of miR-181a on the expression of reporter genes containing wt-XIST or mut-XIST sequences. ** vs Blank, p < 0.01. c Relative expression of XIST in HCC cells and the normal liver cells. ** vs L-02, p < 0.01. d Relative expression of XIST in HCCLM3 cells after transfection with miR-181a inhibitor. ** vs Blank, p < 0.01. e Relative expression of miR-181a in Huh7 cells after transfection with si-XIST or si-Scramble. ** vs Blank, p < 0.01. f Relative expression of miR-181a in HCC clinical samples and the normal. g The relationship between relative expression of XIST and miR-181a. lncRNA, long non-coding RNA; mut, mutant; wt, wild-type; HCC, hepatocellular carcinoma

Mentions: Recent studies showed that microRNA-92b can promote HCC progression by targeting Smad7, mediated by lncRNA XIST [18]. So we explored whether the regulation on PTEN by miR-181a is regulated by lncRNA. Using bioinformatics analysis, we found binding sites between miR-181a and lncRNA XIST, and thus carried out the follow-up study. To explore the interaction between XIST and miR-181a, we constructed three reporter plasmids separately containing one predicted miR-181a binding site on the mRNA of XIST, and three corresponding reporter plasmids with mutant miR-181a binding sites (Fig. 6a). The results of the three binding sites were same (data not shown) so one binding site was selected for further study. Luciferase reporter gene assay showed that miR-181a could significantly inhibit the reporter activities of wt-XIST but not mut-XIST (Fig. 6b). We subsequently detected the expression of XIST in normal liver cells L-02 and HL-7702, and in HCC cells HCCLM3 and Huh7. The results showed the level of XIST in normal liver cells was markedly higher than that in HCC cells. Moreover, the level in Huh7 cells was significantly higher than that in HCCLM3 cells (Fig. 6c). Transfection was used to change the level of XIST in HCC cells. The XIST level in Huh7 cells decreased significantly after transfection with si-XIST (Additional file 4: Figure S2A), whereas XIST level in HCCLM3 cells increased markedly after transfection with pcDNA-XIST (Additional file 4: Figure S2B). Figure 6d shows that the level of XIST in the miR-181a inhibitor group increased significantly compared with both the blank and the inhibitor NC group. The relative expression of miR-181a increased markedly in si-XIST group compared with the blank group (Fig. 6e). Then, we used RT-PCR to determine the relative expression of XIST and the results showed the level in HCC tissues was lower than that in normal tissues (Fig. 6f). The relationship between the relative expression of XIST and miR-181a was determined, and the results showed that the level of XIST was inversely proportional to the miR-181a level (Fig. 6g).Fig. 6


Long non-coding RNA XIST regulates PTEN expression by sponging miR-181a and promotes hepatocellular carcinoma progression
miR-181a is regulated by lncRNA XIST. a Diagram of the three miR-181a putative binding sites and corresponding mutant sites in XIST mRNA sequences. b Effects of miR-181a on the expression of reporter genes containing wt-XIST or mut-XIST sequences. ** vs Blank, p < 0.01. c Relative expression of XIST in HCC cells and the normal liver cells. ** vs L-02, p < 0.01. d Relative expression of XIST in HCCLM3 cells after transfection with miR-181a inhibitor. ** vs Blank, p < 0.01. e Relative expression of miR-181a in Huh7 cells after transfection with si-XIST or si-Scramble. ** vs Blank, p < 0.01. f Relative expression of miR-181a in HCC clinical samples and the normal. g The relationship between relative expression of XIST and miR-181a. lncRNA, long non-coding RNA; mut, mutant; wt, wild-type; HCC, hepatocellular carcinoma
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Fig6: miR-181a is regulated by lncRNA XIST. a Diagram of the three miR-181a putative binding sites and corresponding mutant sites in XIST mRNA sequences. b Effects of miR-181a on the expression of reporter genes containing wt-XIST or mut-XIST sequences. ** vs Blank, p < 0.01. c Relative expression of XIST in HCC cells and the normal liver cells. ** vs L-02, p < 0.01. d Relative expression of XIST in HCCLM3 cells after transfection with miR-181a inhibitor. ** vs Blank, p < 0.01. e Relative expression of miR-181a in Huh7 cells after transfection with si-XIST or si-Scramble. ** vs Blank, p < 0.01. f Relative expression of miR-181a in HCC clinical samples and the normal. g The relationship between relative expression of XIST and miR-181a. lncRNA, long non-coding RNA; mut, mutant; wt, wild-type; HCC, hepatocellular carcinoma
Mentions: Recent studies showed that microRNA-92b can promote HCC progression by targeting Smad7, mediated by lncRNA XIST [18]. So we explored whether the regulation on PTEN by miR-181a is regulated by lncRNA. Using bioinformatics analysis, we found binding sites between miR-181a and lncRNA XIST, and thus carried out the follow-up study. To explore the interaction between XIST and miR-181a, we constructed three reporter plasmids separately containing one predicted miR-181a binding site on the mRNA of XIST, and three corresponding reporter plasmids with mutant miR-181a binding sites (Fig. 6a). The results of the three binding sites were same (data not shown) so one binding site was selected for further study. Luciferase reporter gene assay showed that miR-181a could significantly inhibit the reporter activities of wt-XIST but not mut-XIST (Fig. 6b). We subsequently detected the expression of XIST in normal liver cells L-02 and HL-7702, and in HCC cells HCCLM3 and Huh7. The results showed the level of XIST in normal liver cells was markedly higher than that in HCC cells. Moreover, the level in Huh7 cells was significantly higher than that in HCCLM3 cells (Fig. 6c). Transfection was used to change the level of XIST in HCC cells. The XIST level in Huh7 cells decreased significantly after transfection with si-XIST (Additional file 4: Figure S2A), whereas XIST level in HCCLM3 cells increased markedly after transfection with pcDNA-XIST (Additional file 4: Figure S2B). Figure 6d shows that the level of XIST in the miR-181a inhibitor group increased significantly compared with both the blank and the inhibitor NC group. The relative expression of miR-181a increased markedly in si-XIST group compared with the blank group (Fig. 6e). Then, we used RT-PCR to determine the relative expression of XIST and the results showed the level in HCC tissues was lower than that in normal tissues (Fig. 6f). The relationship between the relative expression of XIST and miR-181a was determined, and the results showed that the level of XIST was inversely proportional to the miR-181a level (Fig. 6g).Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: Tumor metastasis often occurs in hepatocellular carcinoma (HCC) and influences the patient&rsquo;s prognosis, and microRNAs are reported to play key roles in tumor metastasis. This study was conducted to explore the effect of microRNAs on HCC metastasis.

Methods: The levels of miR-181a in HCC tissues, adjacent tissues, metastatic HCC tissues, and non-metastatic HCC tissues at different stages were determined by qRT-PCR. Effect of miR-181a on the proliferation, invasion, and metastasis of HCC cells was estimated by cell counting kits-8 (CCK-8), wound-healing, and Transwell assays. Software analysis and luciferase assays were used to explore the target gene of miR-181a.

Results: MiR-181a was up-regulated in HCC tissues and its expression level in metastatic HCC tissues was much higher than in non-metastasis samples. PTEN was found to be a target gene of miR-181a. MiR-181a had multiple binding sites with the long non-coding RNA (lncRNA) XIST. The regulation of miR-181a on PTEN was mediated by lncRNA XIST. The proliferation and invasion of cells with siXIST were significantly enhanced compared with those of control cells, while knockdown of miR-181a abolished the enhancing effects.

Conclusions: MiR-181a can promote HCC metastasis by targeting PTEN, which is regulated by lncRNA XIST.

Electronic supplementary material: The online version of this article (doi:10.1186/s12885-017-3216-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus